Contrast media for angiography

ABSTRACT

The present invention provides basic ionic contrast agents that have anticoagulant activity. The contrast media incorporate a lysine or arginine group or derivative, and have a free amino or guanidino group. Methods of using the contrast media are also disclosed.

[0001] This application claims priority from provisional applicationSerial No. 60/115,586, filed Jan. 12, 1999, the disclosure of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention concerns compounds useful as contrast mediafor angiography, formulations thereof, and methods of using the same.The present invention is particularly concerned with contrast media thathave anticoagulant properties.

BACKGROUND OF THE INVENTION

[0003] The occurrence of thrombosis during angiographic procedures isproblematic. The anticoagulant properties of the currently availablecommercial contrast media are questionable. The nonionic contrast mediaare being investigated for their potential role in a thrombotic eventduring angiography. The ionic contrast media are thought to have someanticoagulant properties, however, they are less toleratedphysiologically by the patient compared to nonionic contrast media.Accordingly, the provision of a contrast media with both anticoagulantproperties and good physiological properties would be extremelyvaluable.

[0004] U.S. Pat. No. 3,953,501 to Klieger describes compounds of theformula:

[0005] U.S. Pat. No. 4,264,572 to Klieger et al. describes X-Raycontrast media having the following general formula:

[0006] P. Dawson, Embolic Problems in Angiography, Seminars inHematology 28, 31-37 (1991), states that anticoagulant activity can notbe provided in a non-ionic contrast agent without a general increase intoxicity of that agent.

SUMMARY OF THE INVENTION

[0007] The present invention provides basic ionic contrast agents thathave anticoagulant activity.

[0008] A first aspect of the present invention is a compound of FormulaI:

[0009] wherein:

[0010] R1 is selected from the group consisting of alkyl and alkylsubstituted with hydroxy or alkoxy;

[0011] R2, R3 and R4 are each independently selected from the groupconsisting of hydrogen and alkyl;

[0012] R5 and R6 are each independently selected from the groupconsisting of hydrogen, alkyl and hydroxyalky,

[0013] n is from 1 to 3;

[0014] Z is -A-NHCH3, or when R1 is hydroxy or alkoxy substituted alkyland/or when R3 is loweralkyl, Z can also be hydroxy-C2-5-alkylamino;

[0015] A is:

[0016]  wherein

[0017] R7 is H or alkyl;

[0018] R8 is H, alkyl, or alkyl substituted by hydroxy or alkoxy;

[0019] or together R7 and R8 form a propylene or hydroxypropylene ring;

[0020] m is 0 or 1;

[0021] R9 and R10 are each independently H or alkyl; or R9 and R10together form C4-C8 (preferably C4) alkylene which is unsubstituted orsubstituted from one to three times with alkyl (e.g., methyl) orhydroxy;

[0022] R11 is amino or guanidino;

[0023] o is from two to six, preferably 3 or 4;

[0024] or a physiologically acceptable salt thereof.

[0025] A second aspect of the present invention is compounds Formula II:

[0026] wherein:

[0027] R1 is selected from the group consisting of alkyl and alkylsubstituted with hydroxy or alkoxy;

[0028] R2 is selected from the group consisting of hydrogen and alkyl;

[0029] R5 and R12 are each independently selected from the groupconsisting of hydrogen, alkyl and hydroxyalky,

[0030] R9 and R10 are each independently H or alkyl; or R9 and R10together form C4-C8 (preferably C5) alkylene which is unsubstituted orsubstituted from one to three times with alkyl (e.g., methyl) orhydroxy;

[0031] R11 is amino or guanidino;

[0032] o is from two to six, preferably 3 or 4

[0033] or a physiologically acceptable salt thereof.

[0034] A third aspect of the present invention is a contrast mediacomprising an amount of a compound of Formula I or II above effective asa contrast medium in combination with a pharmaceutically acceptablecarrier.

[0035] A fourth aspect of the present invention is the use of a compoundof Formula I or II above for the preparation of a contrast media.

[0036] A fifth aspect of the present invention is a method ofvisualizing an internal organ or structure of a patient which comprisesadministering to the patient an amount of a compound of Formula I or IIabove effective as a contrast medium, and then exposing said organ orstructure to a diagnostic imaging treatment. The structure to be imagedcan be a platelet or fibrin thrombus.

[0037] The present invention is explained in greater detail in thespecification set forth below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0038] Organs or structures that may be imaged by the contrast media ofthe present invention include blood vessels, a thrombus within a vessel,heart, brain, kidney, liver, lungs, spleen, etc. as well as portionsthereof. Thus the entirety of an organ or structure need not be imaged,but only the portion thereof of diagnostic interest.

[0039] Diagnostic imaging treatments used to carry out the method of thepresent invention may be of any type, including magnetic resonanceimaging and CT, but typically are X-ray diagnostic imaging treatments.

[0040] Alkyl as used herein is C1-4 loweralkyl; alkoxy as used herein isC1-C4 loweralkoxy, and more preferably C1-2 loweralkoxy.

[0041] The lower alkyl residue R1, which where appropriate can besubstituted singly or multiply, may contain from 1 to 4 carbon atoms,preferably 1 to 2 carbon atoms, in the alkyl residue. Suitablesubstituents include hydroxy, preferably 1-2 groups, or alkoxy of 1-2carbon atoms, preferably one group. Preferred lower alkyl residues R1have 1-2 carbon atoms and may be substituted by hydroxy or alkoxy, suchas, e.g., methyl, ethyl, methoxymethyl, hydroxymethyl and the like.

[0042] Suitable lower alkyl residues for R2, R3 and R4 have 1-4 carbonatoms, preferably 1-2 carbon atoms, for instance methyl, ethyl, propyl,isopropyl and the like.

[0043] Suitable lower alkyl residues for R5 have 1-4 carbon atoms andespecially have 1-2 carbon atoms, for instance, methyl or ethyl. Thisalkyl residue may optionally be substituted by hydroxy, preferably 1-2groups; n preferably has a value of 1.

[0044] When Z is straight-chain or branched hydroxy lower alkylamino,the alkyl residue may contain 2-5 carbon atoms, and, e.g., 1-3 hydroxysubstituents. If Z is a straight chain group, the alkyl residuepreferably contains 2-5 carbon atoms; if Z is branched, the alkylresidue preferably contains 3-5 carbon atoms. The hydroxy groups in Zmay be present as primary or secondary hydroxy groups. Suitable Zresidues include, e.g., 2-hydroxypropylamino, 3-hydroxypropylamino,2-hydroxy-1, 1-dimethylethylamino, 3-hydroxy-1, 1-dimethylpropylaminoand, preferably, 2-hydroxyethylamino.

[0045] Suitable bridging amino acid residues A include those derivedfrom an amino carboxylic acid of arbitrary configuration. Included arethose residues which are equivalents of, e.g., the preferred amino acidresidue of the formula

[0046] wherein m is 0 or 1, R7 is hydrogen or lower alkyl of 1-4 carbonatoms, preferably 1-2 carbon atoms, R8 is hydrogen or lower alkyl of 1-4carbon atoms which also may be straightchain or branched and which maybe substituted by hydroxy, preferably 1-2 groups or lower alkoxy of 1-2carbon atoms, preferably 1-2 groups, and wherein R7 and R8 together mayform a propylene or hydroxypropylene group. Examples of suitableaminocarboxylic acids include glycine, alanine, valine, serine,O-methylserine, proline, hydroxyproline, leucine, isoleucine, sarcosine,beta-alanine and the like. Especially preferred arealpha-amino-carboxylic acids in which R7 is hydrogen or methyl and R8 ishydrogen or lower alkyl of 1-2 carbon atoms which may be hydroxy- ormethoxy-substituted.

[0047] Compounds of Formula I are prepared as described in Examples 1-5below, or variations thereof that will be apparent to those skilled inthe art. Examples of such compounds include the following:

[0048] Compounds of Formula II are prepared as described in Examples 1and 6-8 below, or variations thereof that will be apparent to thoseskilled in the art.

[0049] Additional examples of compounds of the invention include thefollowing:

[0050] Additional examples of compounds of the invention include thefollowing:

[0051] Additional examples of compounds of the invention include thefollowing, where R groups are as defined in the Table below:

where X is as defined above.   R₂ R₃ 1 CONHCH(CH₂CH)₂ NHCOCH(OH)CH₃ 2CONHCH₂CH(OH)CH₂OH N(COCH₃)CH₂CH(OH)CH₂OH 3 CONHCH₂CH(OH)CH₂OHN(COCH₂OH)CH₂CH(OH)CH₂OH 4 CONHCH₂CH₂OH N(COCH₃)CH₂CH(OH)CH₂OH 5CON(CH₃)CH₂CHOHCH₂OH NHCOCH₂OCH₃

[0052] Still additional examples of compounds of the present inventioninclude the following: Base (Prior Art) Compoun¹:

Where X is as define above   R₁ R₂ 1 N(CH₃CH₂OH)CH₂CH(OH)CH(OH)CH₂OHCH(OH)CH₂OH 2 N(CH₃CH₂OH)CH₂CH(OH)CH(OH)CH₂OH CH(CH₂OH)₂ 3N(CH₂CH(OH)CH₂OH)CH₂CH(OH)CH(OH)CH₂OH CH(OH)CH₃

[0053]

[0054] The foregoing compounds are made in accordance with theprocedures described herein, or variations thereof that will be apparentto those skilled in the art in light of the specific compound desired.

[0055] To form salts of the foregoing compounds, any of the conventionalphysiologically acceptable counterions are suitable. In general, saltsof the active compound may have the general formula R⁺X⁻, where R⁺ isthe active compound as described above and X⁻ is Cl⁻, HCOO⁻, H₂citrate,CH₃COO, NH₂CH₂COO⁻, HCO₃ ⁻, H₂PO₄ ⁻, or HSO₃ ⁻.

[0056] As noted above, the new compounds of this invention are useful ascontrast media, and are especially suited for use in urography,angiography, bronchography, etc., for visualization of body cavities andalso for computer tomography.

[0057] B. Formulations and Administration

[0058] The preparation and administration of new contrast media from thecompounds of this invention is in accordance with known techniques. Forexample, the contrast compound can be combined with conventional galenicadjuvants to form a composition suitable for the desired method ofapplication.

[0059] For example, the compounds of this invention can be employed inmixture with conventional excipients, i.e., pharmaceutically acceptableorganic or inorganic carrier substances suitable for parenteral orenteral application which do not deleteriously react with the activecompounds. Suitable pharmaceutically acceptable carriers include but arenot limited to water, salt solutions, alcohols, vegetable oils,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, talc, etc.

[0060] For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions, as well as suspensions oremulsions. Ampoules are convenient unit dosages.

[0061] For enteral application, particularly suitable are tablets ordragees having the talc and/or a carbohydrate carrier or binder or thelike, the carrier preferably being lactose and/or corn starch and/orpotato starch. A syrup or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can also beformulated wherein the active compound is protected with differentiallydegradable coatings, e.g., by microencapsulation, multiple coatings,etc.

[0062] The concentration of the new contrast media of this invention inaqueous media depends on the particular diagnostic method involved. Thepreferred concentrations and doses of the compounds of this invention,e.g., for X-ray diagnoses, are concentrations of 50-400 mg of iodine perml and doses of 10-500 ml. Concentrations of 100-350 mg of iodine per mlare especially preferred.

[0063] The precise method and details of application depend on the organwhich is to be visualized and can be determined by fully conventionalconsiderations, e.g., in analogy with conventional media such as thosedescribed in U.S. Pat. No. 4,264,572.

[0064] In the Examples below, DMAP means dimethylaminopyridine, DMAcmeans dimethylacetamide, BOC means t-butylcarbamate; DMF meansdimethylformamide; nMM means N-methylmorpholine; nMP meansN-methylpiperidine; 4MP means 4-methylpiperidine; iBCF meansisobutylchloroformate; DIEA means diethylamine; THF meanstetrahydrofuran; Fmoc means 9-fluorenylmethoxycarbonyl; Lys meanslysine; AcOH means acetic acid AcOEt means ethyl acetate, NMR meansNuclear Magnetic Resonance Spectroscopy, TLC means thin layerchromatography N means Normal, h means hour, min. means minutes, RTmeans room temperature, and temperatures are given in degreescentigrade.

EXAMPLE 1 Preparation of Lys(Boc)4MP

[0065] This example describes the preparation of the intermediateLys(Boc)4MP. The reaction schemes are outlined below. In reaction 1,step 1, a solution of FmocLys(Boc) (1 Equiv) and nMM (1 Equiv) in THF iscooled to 0° C. and iBCF (1 Equiv) is added. In step 2, after 15 minutes4-methylpiperidine (1 Equiv) is added and the mixture is maintained at0° C. for 10 minutes and then allowed to warm to room temperature for 40minutes. In step 3, the reaction mixture is concentrated under reducedpressure and extracted with EtOAc; washed (0.5 N KHCO₃, 0.5 N HCl) andevaporated. TLC is performed to verify reaction components.

[0066] In reaction 2, step 1, the crude FmocLys(Boc)4MP is dissolved inTHF and DIEA (9/1, v/v) and allowed to stand at RT for 2 h. In step 2,the reaction mixture is concentrated under reduced pressure andtriturated with ether/hexane (4/1, v/v). TLC is performed as well as NMRand Mass Spectrometry.

EXAMPLES 2-5 Preparation of Compound 1

[0067] Examples 2-5 illustrate the preparation of Compound 1 above,which is illustrative of Formula I above.

EXAMPLE 2 Acetate Protection of the Hydroxyl Group

[0068] The compoundN-(2-hydroxyethyl)-2,4,6-triiodo-5-[2-(2,4,6triiodo-3-(N-methylacetamido)-5-(methylcarbamyl)benzamidoacetamido]-isophthalmic acid, ioxaglate (CAS 59017-64-0) is placed intoa 3-necked round bottom flask equipped with a mechanical stirrer anddropping funnel. A catalytic amount of DMAP is added. DMAc is then addedto the flask. Next, acetic anhydride (1.6 Equiv) is added dropwise andthe mixture stirred at room temperature overnight. Finally, water isadded and the mixture filtered, collecting the product. The solidproduct is dried in a vacuum oven at 70° C. for 3 days prior to use.

EXAMPLE 3 Acid Chloride Formation

[0069] In step 1, the product of Example 2 is placed in a 3-necked roundbottom flask equipped with a mechanical stirrer, a dropping funnel and acondenser.

[0070] In step 2, thionyl chloride (16 Equiv.) is rapidly added to theflask via the dropping funnel. The solution is heated to 80-85° C. withan oil bath for three hours.

[0071] In step 3, check for product by thin layer chromatography (100%ethyl acetate).

[0072] In step 4, the condenser is replaced with a distilling arm.Excess thionyl chloride is removed by distillation under house vacuum(oil bath temperature 50° C.).

[0073] In step 5, THF is added and distilled. The addition/distillationis repeated twice.

[0074] In step 6, the product (AcHexCl) is extracted as an oil withethyl acetate, washing with saturated NaHCO₃ and NaCl. The product isdried over Na₂SO₄, filtered and concentrated under reduced pressure.

EXAMPLE 4 Amide Coupling Reaction

[0075] In step 1, the product of Example 3 (AcHexCl) and sodiumcarbonate (1 Equiv) is placed in a round bottom flask equipped with amagnetic stir bar. Anhydrous DMAc is added and stirred under nitrogenfor five minutes.

[0076] In step 2, the product of Example 1 is added (41% w/v in DMAc,1.5 Equiv.) via a syringe to the flask. The contents are stirredovernight under nitrogen at room temperature.

[0077] In step 3, the reaction mixture is filtered through celite andthe DMAc is removed by distillation under high vacuum.

[0078] In step 4, methanol is added to dissolve the oil and then wateris added. The reaction solution is passed through an IRA-120H resin andthe eluent collected. The resin is washed with water/methanol (1:1, v/v)and the eluent washes combined. The solution is then used for the acidicdeprotection of the compound.

EXAMPLE 5 Acidic Deprotection

[0079] The product of Example 4 is deprotected to yield Compound 1illustrated below as follows.

[0080] The product of Example 4 is acidified to pH 1.0 in methanol/watersolution with concentrated H₂SO₄.

[0081] The mixture is then heated with an oil bath to reflux for 2 hoursand the product concentrated.

[0082] Water is then added and the mixture is heated to reflux for anadditional two hours, and the product is concentrated.

[0083] Water is then added and the mixture heated to reflux with a DeanStark trap. The distillate is collected in the trap and allowed to coolto room temperature overnight.

[0084] The solution is subjected to TLC with EtOAc/Methanol/AcOH, 10:5:1v/v/v.

[0085] The filtrate is then placed on an exchange column (AmberliteIRA-68 weakly basic anion exchange resin on top of IRA-458 stronglybasic anion exchange resin) and the filtrate collected and the columnwashed with deionized water.

[0086] The filtrate is concentrated under reduced pressure and thenunder high vacuum with the flask warmed at 50° C. in a water bathovernight.

EXAMPLES 6-8 Preparation of Compound 10

[0087] These examples describe the preparation of Compound 10, which isillustrative of Formula II above.

EXAMPLE 6 Acid Chloride Formation

[0088] Thionyl chloride (16 Equiv.) is added dropwise to a solution ofiothalamic acid (CAS 2276-90-6). The solution is heated and stirred for3 hours. After removal of excess thionyl chloride by distillation, theproduct is extracted and concentrated under reduced pressure.

EXAMPLE 7 Amide Coupling

[0089] Lys(Boc)4MP prepared as described in Example 1 (1.5 Equiv.) isadded to a sodium carbonate solution of the acid chloride under nitrogenand stirred overnight at room temperature. The reaction mixture isfiltered, concentrated, and extracted to yield the protected amideintermediate illustrated below.

EXAMPLE 8 Acidic Deprotection

[0090] The intermediate prepared in example 7 is dissolved intrifluoroacetic acid and dichloromethane (1:1 v/v) and allowed to standat room temperature for 1 hour. The reaction mixture is concentratedunder reduced pressure and the product is crystallized to yield Compound10 shown below.

EXAMPLE 9 Formulations and Administration of Active Compound

[0091] Compound 1 and Compound 10 as described above are prepared as aformulation as follows:

[0092] 320 mg Iodine/ml (420 mM of compound 1 and 845 mM of compound 10)is pH adjusted to 7.4 to 8.8 with NaOH or HCl in an aqueous solutionconsisting of 10 mM tromethane (Tris), 0.10 mg/ml edetate calciumdisodium, 19 mM NaCl, and 0.3 mM Calcium chloride, dihydrate. Thisformulation is used in the dose regimens described below.

[0093] Selective Coronary Arteriography with or without LeftVentriculography

[0094] The usual dose for left coronary is 2-14 mL (typically 8 mL) ofthe formulation described above, and the usual dose for right coronaryarteriography is 1 to 10 mL (typically 5 mL) of the formulationdescribed above. The doses may be repeated as necessary. Doses up to atotal of 150 mL are suitable. For left ventriculography, the usual dosein a single injection is 35-45 mL (typically 45 mL) and repeated asnecessary. The total dose for combined selective coronary arteriographyand left ventriculography should not exceed 250 mL.

[0095] Peripheral Arteriography

[0096] The usual single adult dose for aorto-iliac runoff studies is 20to 80 mL (typically 45 mL). The usual single adult dose for the commoniliac, the external iliac and the femoral arteries is 10-50 mL(typically 30 mL). These doses may be repeated as necessary. For theupper limb, the usual single adult dose is 20 mL (range 15-30 mL)repeated as necessary. The total procedural dose should not exceed 250mL.

[0097] Aortography and Selective Visceral Arteriography

[0098] The usual dose for injections into the aort is 25 to 50 mL; theusual dose for injection into the celiac artery is 40 mL; the usual dosefor injection into the superior mesenteric artery is 20 to 40 mL; theusual dose for injection into the inferior mesenteric artery is 8 to 15mL. These doses may be repeated as necessary. The total dose should notexceed 250 mL.

[0099] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

We claim:
 1. A compound of the formula:

wherein: R1 is selected from the group consisting of alkyl and alkylsubstituted with hydroxy or alkoxy; R2, R3 and R4 are each independentlyselected from the group consisting of hydrogen and alkyl; R5 and R6 areeach independently selected from the group consisting of hydrogen, alkyland hydroxyalky, n is from 1 to 3; Z is -A-NHCH3, or when R1 is hydroxyor alkoxy substituted alkyl and/or when R3 is loweralkyl, or Z ishydroxy-C2-5-alkylamino; A is:

 wherein R7 is H or alkyl; R8 is H, alkyl, or alkyl substituted byhydroxy or alkoxy; or together R7 and R8 form a propylene orhydroxypropylene ring; m is 0 or 1; R9 and R10 are each independently Hor alkyl; or R9 and R10 together form C4-C8 alkylene which isunsubstituted or substituted from one to three times with alkyl orhydroxy; R11 is amino or guanidino; o is from two to six; or aphysiologically acceptable salt thereof.
 2. A compound according toclaim 1, wherein R3 and R4 are each hydrogen.
 3. A compound according toclaim 1, wherein R₁₂ is hydrogen and R₅ is alkyl.
 4. A compoundaccording to claim 1, wherein n is
 1. 5. A compound according to claim1, wherein R1 is alkyl.
 6. A compound according to claim 1, wherein R2is alkyl.
 7. A compound according to claim 1, wherein Z is —NCH₂CH₂OH.8. A compound according to claim 1, wherein o is
 4. 9. A compoundaccording to claim 1, wherein R₁₁ is amino.
 10. A compound according toclaim 1, wherein R₉ and R₁₀ together form C5 alkylene, which alkylene isunsubstituted or substituted once with alkyl.
 11. A compound of theformula:

wherein: R1 is selected from the group consisting of alkyl and alkylsubstituted with hydroxy or alkoxy; R2 is selected from the groupconsisting of hydrogen and alkyl; R5 and R6 are each independently isselected from the group consisting of hydrogen, alkyl and hydroxyalky,R9 and R10 are each independently H or alkyl; or R9 and R10 togetherform C4-C8 alkylene which is unsubstituted or substituted from one tothree times with alkyl or hydroxy; R11 is amino or guanidino; o is fromtwo to six; or a physiologically acceptable salt thereof.
 12. A compoundaccording to claim 11, wherein R₁ is alkyl, which is unsubstituted orsubstituted with hydroxy.
 13. A compound according to claim 11, whereinR₂ is hydrogen.
 14. A compound according to claim 11, wherein R₁₂ ishydrogen and R₅ is alkyl.
 15. A compound according to claim 11, whereino is
 4. 16. A compound according to claim 1, wherein R₁₁ is amino.
 17. Acompound according to claim 1, wherein R₉ and R₁₀ together form C5alkylene, which alkylene is unsubstituted or substituted once withalkyl.